The initiation of sporulation and the production of antibiotics in Gram-positive sporulating soil bacteria such as Bacillus subtilis occurs in response to nutrient limitation. The expression of many carbon and nitrogen degradative pathways is also regulated in response to nutrient availability in B. subtilis. Our long term objective is to understand the network of metabolic signals and systems regulating the utilization of nitrogen compounds in this bacterium. The genes involved in nitrogen metabolism in B. subtilis are controlled by three systems, each of which regulates an unique subset of genes in response to different nutritional conditions. TnrA regulation occurs during nitrogen-limited growth. GlnR, the TnrA homolog, functions as a regulatory protein in cells grown with excess nitrogen. CodY controls gene expression in response to growth rate. In this proposal, biochemical and genetic approaches will be used to investigate the molecular mechanism by which TnrA regulates gene expression. The interaction of the purified TnrA protein with wild-type and mutant TnrA sites will be examined in in vitro DNA binding experiments and using an in vitro transcription system. The wild-type B. subtilis glutamine synthetase (GS) protein is required for signal transduction to the TnrA protein. The ability of the wild-type B. subtilis GS protein to regulate the TnrA regulatory activity will be examined in a heterologous system, Escherichia coli cells. trans-acting factors involved in TnrA regulation will be sought by isolating mutants with altered regulation of nrgAB expression. DNA complementing these mutants will be cloned and sequenced. The GlnR/TnrA sites regulating the transcription of the glnRA, tnrA and ureABC P3 promoters by TnrA and GlnR will be identified genetically and verified in in vitro DNA binding experiments. Additional genes in the TnrA regulon will be sought by determining whether TnrA is required for the nitrogen regulation of two asparaginase genes, ansB and ansZ. These studies should help define the novel paradigms regulating gene expression in Gram-positive soil bacteria. In addition, they will provide both information and genetic tools for optimizing the production of medically and agriculturally important compounds in Bacilli spp. by commercial fermentation or during growth in the soil, where nutrients are relatively scarce.